Ethanol (CH3CH2OH) can be used as a liquid fuel to run engines such as cars. A significant market for ethanol as a liquid fuel already exists in the current transportation and energy systems. In the United States, currently, ethanol is generated primarily from corn starch using a yeast-fermentation process. Therefore, the “cornstarch ethanol production” process requires a number of energy-consuming steps including agricultural corn-crop cultivation, corn-grain harvesting, corn-grain starch processing, and starch-to-sugar-to-ethanol fermentation. Independent studies have recently shown that the net energy gain of the “cornstarch ethanol production” process is very limited. That is, the “cornstarch ethanol production” process costs nearly as much energy as the energy value of its product ethanol. This is not surprising, understandably because the cornstarch that the current technology can use represents only a small fraction of the corn crop biomass that includes the corn stalks, leaves and roots. The cornstovers are commonly discarded in the agricultural fields where they slowly decompose back to CO2, because they represent largely lignocellulosic biomass materials that the current biorefinery industry cannot efficiently use for ethanol production. There are research efforts in trying to make ethanol from lignocellulosic plant biomass materials—a concept called “cellulosic ethanol”. However, plant biomass has evolved effective mechanisms for resisting assault on its cell-wall structural sugars from the microbial and animal kingdoms. This property underlies a natural recalcitrance, creating roadblocks to the cost-effective transformation of lignocellulosic biomass to fermentable sugars. Therefore, one of its problems known as the “lignocellulosic recalcitrance” represents a formidable technical barrier to the cost-effective conversion of plant biomass to fermentable sugars. That is, because of the recalcitrance problem, lignocellulosic biomasses (such as cornstover, switchgrass, and woody plant materials) could not be readily converted to fermentable sugars to make ethanol without certain pretreatment, which is often associated with high processing cost. Despite more than 50 years of R&D efforts in lignocellulosic biomass pretreatment and fermentative ethanol-production processing, the problem of recalcitrant lignocellulosics still remains as a formidable technical barrier that has not yet been eliminated so far. Furthermore, the steps of lignocellulosic biomass cultivation, harvesting, pretreatment processing, and cellulose-to-sugar-to-ethanol fermentation all cost energy. Therefore, any new technology that could bypass these bottleneck problems of the biomass technology would be useful.
Oxyphotobacteria are prokaryotic organisms that are capable of performing oxygenic autotrophic photosynthesis using water as the source of electrons and carbon dioxide as the source of carbon. In nature, there are two orders of oxygenic photosynthetic prokaryotes within the class of the Oxyphotobacteria: Cyanobacteria (such as, Synechococcus elongatus, Anabaena sp., Synechocvstis sp., Nostoc punctiforme, Spirulina platensis, and Thermosynechococcus elongatus) and Oxychlorobacteria (such as Prochlorococcus marinus, Prochloron didemni, and Prochlorothrix hollandica). Cyanobacteria are commonly also known as “blue-green algae”; and Oxychlorobacteria are sometimes regarded as “the ‘other’ Cyanobacteria” or more scientifically classified as Prochlorophytes since they contain both chlorophyll-a (Chl-a) and chlorophyll-b (Chl-b). For example, Prochlorococcus marinus MED4 (oxychlorobacterium) possesses an unorthodox pigment composition of divinyl derivatives of Chl-a and Chl-b, a-carotene, zeaxanthin, and a type of phycoerythrin. By contrast, the highly related Synechococcus (cyanobacterium) contains Chl-a and phycobilins that are more typical of cyanobacteria. However, both Cyanobacteria and Oxychlorobacteria can perform photosynthetic assimilation of CO2 with 0, evolution from water in a liquid culture medium with a maximal theoretical solar-to-biomass energy conversion of about 10%; these oxygenic photosynthetic prokaryotes have tremendous potential to be a clean and renewable energy resource. However, the wild-type oxygenic photosynthetic organisms, such as the wild-type cyanobacteria, do not possess the ability to produce ethanol directly from CO2 and H2O. The wild-type photosynthesis uses the reducing power (NADPH) and energy (ATP) from the photosynthetic water splitting and proton gradient-coupled electron transport process through the thylakoid membrane system to reduce CO2 into carbohydrates (CH2O)n with a series of enzymes collectively called the “Calvin cycle” at the cytoplasm stroma region in Cyanobacteria. The net result of the wild-type photosynthetic process is the conversion of CO2 and H2O into carbohydrates (CH2O)n and O2 using sunlight energy according to the following process reaction:nCO2+nH2O→(CH2O)n+nO2  [1]The carbohydrates (CH2O)n are then further converted to all kinds of complicated cellular (biomass) materials including proteins, lipids, glycogen, and cellulose and other cell-structural materials during cell metabolism and growth.
Based on the current scientific knowledge, wild-type oxyphotobacteria including cyanobacteria (such as Synechococcus sp. PCC 7942, Nostoc sp. PCC 7120, Synechomtis sp. PCC 6803, and Thermosynechococcus elongatus BP-1) and oxychlorobacteria (such as Prochlorococcus marinus MIT 9313, Prochlorococcus marinus SS120, and Prochlorococcus marinus MED4) are not capable of photosynthetic ethanol production directly from CO2 and H2O. The fundamental properties of oxygenic photosynthesis in oxyphotobacteria are quite similar to those in eukaryotic algae and higher plants. However, there are also some significant differences between the prokaryotes (oxyphotobacteria) and the eukaryotes (algae and higher plants). The Calvin-cycle activity (the photosynthetic CO2-fixation process) in eukaryotic algae (and higher plants) occurs inside a chloroplast, which is a well-organized photosynthetic organelle. On the other hand, the Calvin-cycle activity in oxyphotobacteria occurs in the cytoplasm since the prokaryotic organisms do not have a chloroplast organelle. In addition, oxyphotobacteria as prokaryotes do not have a nucleus organelle; and their molecular genetic organization and machineries are also somewhat different from those of the eukaryotes. The present application discloses a more-specific method in creating prokaryotic designer oxyphotobacteria for photosynthetic ethanol production directly from CO2 and H2O.
The present invention provides a photobiological ethanol production and harvesting methodology with greenhouse distillation systems and designer transgenic oxyphotobacteria that are capable of synthesizing ethanol directly from CO2 and H2O.
The integrated photobiological ethanol production and harvesting technology provided by the present invention could bypass all the bottleneck problems of the biomass industry mentioned above.